1. Field of the Invention
The present invention generally relates to methods and apparatus for automatically and rapidly making determinations of the total number of bacteria, alive and dead, present in a fluid sample by using the iron porphyrin assay method. More particularly, the invention relates to methods and apparatus for increasing the reliability of the iron porphyrin assay by inactivating soluble iron porphyrins in the sample which are unrelated to the bacteria in the sample but which would luminescently react with luminol to interfere with the assay.
2. Description of the Prior Art
Prior art methods have recognized and utilized the light reaction of luminol in hydrogen peroxide, together with the ability of microorganisms to decompose the peroxide through the enzyme catalase. For example, Soli, U.S. Pat. No. 3,564,588, has used the foregoing chemiluminescent technique for the detection of living organisms and thereby to differentiate living organisms from inert matter. In Soli's process, it is crucial that there initially be a sustained light reaction provided by combining the peroxide and luminol before adding a fluid sample solution suspected of containing living organisms. If the added solution does contain living organisms, Soli reports that there will be a noticeable decay in the light reactions, which drop in light intensity, in the practice of his process, is interpreted as revealing the presence of the living organisms.
A second approach in the prior art to the rapid identification of bacteria using chemiluminescence is found in Witz et al., U.S. Pat. No. 3,959,081, in which a recording of the decaying light emission, including at least a portion of the light build-up, provides a characteristic time curve which has been found distinctive for each different type of microorganism, and thus, makes it possible to define specific microorganisms contained within the sample. Additionally, it has been found that certain non-biological agents, which may be present in a sample, e.g., Fe.sup.++, Cu.sup.++ and So.sub.2 also react with a luminol reagent to initiate luminescence and have light emission characteristics which are different from most biological agents. The light emission curves of the foregoing-named inorganic agents generally terminate in less than one second and since the reaction time required to reach maximum luminescence of mostmicroorganisms of interest exceeds the one second period, typically five to eight seconds and longer. Thus it is possible to record only a portion of the light emission curves and avoid noise background of the inorganic agents.
However, in both of the above described methods, a number of compounds produce luminescent response curves similar to the curves produced by some microorganisms. Compounds formed from metallic ions such as Ferricyanide and Hypochlorate and a number of chelated transition metals such as Ferrous and Cobaltous ions react with luminol hydrogen peroxide to produce luminescence. When all the necessary luminol reagents are present in excess, the amount of light emitted from the luminol reaction (.lambda..sub.max =425 nm) is proportional to the concentration of such metallic ions and any free porphyrins present as well as such porphyrins as are released from the bacteria present. If, however, only the reaction of luminol with those iron porphyrins released from the bacteria could be measured, the light response could then be related to the number of bacteria present in the sample.
The present invention overcomes the deficiencies of the prior art by providing methods and apparatus for removing luminolreacting interference material prior to measuring the light reaction so that the reaction light relates only to the bacteria present in the sample.